ProjectSummary Amajorchallengeforthecancergenomicscommunityisdeterminingwhichsomaticmutationsarecontributing totumorigenesisandwhichare?passenger?(neutral)mutations.SomaticmutationsthatcausealteredmRNA splicinghaverecentlybeenappreciatedasoncogenicdriverevents,suchasthecasewithskippingofexon14 intheMETproto-oncogene.Additionally,lungcancerpatientswithskippingofMETexon14respondwellto targeteddruginhibitors.ThroughcomputationalanalysisofbothDNAandmRNAsequencingdataof495lung cancerdonorsamples,wehaveidentified635casesofexon-skippingassociatedwithsomaticmutation;? however,welackexperimentalevidencetoknowwhichoftheseexonsmightalsorepresentoncogenicdriver events.Thisprojectwilltestthehypothesisthatasubsetofexon-skippingeventsassociatedwith somaticmutationsarenoveloncogenicalterations.Totestthishypothesis,ourfirstaimwilldetermineif previouslyuncharacterizedexon-skippingevents,associatedwithsomaticmutationsinRASpathwaygenes, areoncogenic.RASpathwaygenesareknowntoberecurrentlymutatedinlungcancerandmanyare targetablealterations;?therefore,wewillfocusourinitialstudiesonexon-skippingeventswhicharemostlikely tobeoncogenicandmostlikelytoleadtoatherapeutictarget.Oursecondaimwilldevelopanovelhigh- throughputCRISPR-Cas9screentargetingall635exonstotestifanyoftheseexonsareoncogenicwhenthey areskipped.Ourfinalaimwillusehomology-directedCRISPR-Cas9toknock-incandidatesplicemutations andvalidatethatthesemutationscauseexon-skipping,whichleadstoanoncogenicgenealteration. Completionofthisstudywillsettheframeworkformorebroadstudiesofsomaticmutationsthataffectgene functionthroughalternativesplicingandtoinvestigatenoveltargetedtherapies.